RESUMO
Rhabdomyosarcoma tumor cells resemble differentiating skeletal muscle cells, which unlike normal muscle cells, fail to undergo terminal differentiation, underlying their proliferative and metastatic properties. We identify the corepressor TLE3 as a key regulator of rhabdomyosarcoma tumorigenesis by inhibiting the Wnt-pathway. Loss of TLE3 function leads to Wnt-pathway activation, reduced proliferation, decreased migration, and enhanced differentiation in rhabdomyosarcoma cells. Muscle-specific TLE3-knockout results in enhanced expression of terminal myogenic differentiation markers during normal mouse development. TLE3-knockout rhabdomyosarcoma cell xenografts result in significantly smaller tumors characterized by reduced proliferation, increased apoptosis and enhanced differentiation. We demonstrate that TLE3 interacts with and recruits the histone methyltransferase KMT1A, leading to repression of target gene activation and inhibition of differentiation in rhabdomyosarcoma. A combination drug therapy regime to promote Wnt-pathway activation by the small molecule BIO and inhibit KMT1A by the drug chaetocin led to significantly reduced tumor volume, decreased proliferation, increased expression of differentiation markers and increased survival in rhabdomyosarcoma tumor-bearing mice. Thus, TLE3, the Wnt-pathway and KMT1A are excellent drug targets which can be exploited for treating rhabdomyosarcoma tumors.
Assuntos
Rabdomiossarcoma , Humanos , Camundongos , Animais , Proteínas Correpressoras/genética , Histona Metiltransferases , Diferenciação Celular/genética , Rabdomiossarcoma/patologia , Antígenos de Diferenciação , Proliferação de Células/genética , Linhagem Celular TumoralRESUMO
The effect of fisetin on autophagy in hepatocellular carcinoma remains uncovered. HepG2 cells were exposed to different concentrations of fisetin (25, 50, and 100 µM) for 24 h. The cells were also treated with rapamycin and chloroquine alone or in combination with fisetin. Autophagic flux formation and ATP levels were determined. The changes in autophagic markers and AMPK signaling proteins were analyzed using qRT-PCR and Western blotting. Cyto-ID staining followed by flow cytometry showed that fisetin decreased autophagic flux formation in a dose-dependent manner. In gene expression analysis, the mRNA levels of mTOR, Atg5, Atg16L, and LC3A were elevated, whereas the mRNA levels of Atg7 and Beclin1 were downregulated in a dose-dependent manner compared to control. In the Western blotting analysis, fisetin treatment inhibited the expression of Atg7, Atg16L, mTOR, and pACC and elevated the expression of Atg5, AMPKα, AMPKß1/2, ACC and Akt. Taken together, the results revealed that fisetin inhibited autophagy by the activation of PI3K/Akt/mTOR and modulation of AMPK signaling pathways. Our findings indicate that suppression of autophagy by fisetin may serve as an effective therapeutic strategy against HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Quinases Ativadas por AMP/genética , Apoptose , Autofagia , Carcinoma Hepatocelular/tratamento farmacológico , Flavonóis , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Fisetin (3,7,3',4'-tetrahydroxyflavone), a bioactive dietary flavonoid, intrigued scientists for its anticancer potential against various cancer types. We investigated the fisetin-induced inhibition of growth and survival of human hepatocellular carcinoma. Fisetin decreased cell viability and proliferation of HepG2 cells as revealed from MTT and clonogenicity assays. Cell cycle arrest in the G2/M phase was observed. Annexin V/propidium iodide (PI) staining followed by flow cytometry revealed that fisetin induced both apoptosis and necroptosis in HepG2 cells. Apoptotic cells were significantly increased on fisetin treatment as observed in morphological evaluations and 4',6-diamidino-2-phenylindole and Acridine orange staining. Flow cytometry, fluorescence imaging, and 2', 7'-dichlorofluorescein diacetate analyses showed an increase in reactive oxygen species (ROS) generation on fisetin treatment. Pretreatment with N-acetyl cysteine inhibited ROS production and also rescued mitochondrial membrane potential in HepG2 cells. The underlying mechanisms of apoptosis and necroptosis were determined by analysis of their respective signaling molecules using qRT-PCR and Western blotting. Fisetin showed a marked increase in the expression of TNFα and IKκB with a decrease in NF-κB, pNF-κB and pIKκB expression. Fisetin reduced the expression of Bcl2, and elevated levels of Bax, caspase-3, and PARP and thus induced apoptosis in HepG2 cells. zVAD suppressed the fisetin-induced expression of caspase-8, RIPK1, RIPK3, and MLKL as opposed to fisetin treatment. Nec-1 + fisetin could not completely block necroptosis, which warrants further investigation. Taken together, our findings demonstrate that the fisetin exhibited anti-proliferative effects on HepG2 cells through apoptosis and necroptosis via multiple signaling pathways. Fiestin has potential as a therapeutic agent against hepatocellular carcinoma.
Assuntos
Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Flavonoides/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Flavonóis , Células Hep G2 , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
In the post-genomic era, deciphering the Nrf2 binding sites - antioxidant response elements (AREs) is an essential task that underlies and governs the Keap1-Nrf2-ARE pathway - a cell survival response pathway to environmental stresses in the vertebrate model system. AREs regulate the transcription of a repertoire of phase II detoxifying and/or oxidative-stress responsive genes, offering protection against toxic chemicals, carcinogens, and xenobiotics. In order to identify and analyze AREs in zebrafish, a pattern search algorithm was developed to identify AREs and computational tools available online were utilized to analyze the identified AREs in zebrafish. This study identified the AREs within 30â¯kb upstream from the transcription start site of antioxidant genes and mitochondrial genes. We report for the first time the AREs of all the known protein coding genes in the zebrafish genome. Western blotting, RT2 profiler array PCR, and qRT-PCR were performed to test whether AREs influence the Nrf2 target genes expression in the zebrafish larvae using sulforaphane. This study reveals unique AREs that have not been previously reported in the cytoprotective genes. Nine TGAG/CNNNTC and six TGAG/CNNNGC AREs were observed significantly. Our findings suggest that AREs drive the dynamic transcriptional events of Nrf2 target genes in the zebrafish larvae on exposure to sulforaphane. The identified abundant putative AREs will define the Keap1-Nrf2-ARE network and elucidate the precise regulation of Nrf2-ARE pathway in not only diseases but also in embryonic development, inflammation, and aerobic respiration. Our results help to understand the dynamic complexity of the Nrf2-ARE system in zebrafish.
Assuntos
Elementos de Resposta Antioxidante/genética , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Isotiocianatos/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfóxidos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Peixe-ZebraRESUMO
With increased industrial utilization of iron oxide nanoparticles (Fe2O3-NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe2O3-NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe2O3-NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe2O3-NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe2O3-NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe2O3-NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe2O3-NPs could be an environmental risk factor for reproductive disease.
Assuntos
Compostos Férricos/toxicidade , Proteínas de Choque Térmico/biossíntese , Nanopartículas Metálicas/toxicidade , Próstata/metabolismo , Proteínas Secretadas pela Próstata/biossíntese , Glândulas Seminais/metabolismo , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Masculino , Camundongos , Próstata/efeitos dos fármacos , Proteínas Secretadas pela Próstata/genética , Distribuição Aleatória , Glândulas Seminais/efeitos dos fármacos , Difração de Raios XRESUMO
Fluorosis is an endemic global problem causing male reproductive impairment. F mediates male reproductive toxicity in mice down-regulating 63 genes involved in diverse biological processes - apoptosis, cell cycle, cell signaling, chemotaxis, electron transport, glycolysis, oxidative stress, sperm capacitation and spermatogenesis. We predicted the miRNAs down-regulating these 63 genes using TargetScan, DIANA and MicroCosm. The prediction tools identified 3059 miRNAs targeting 63 genes. Of the predicted interactions, 11 miRNAs (mmu-miR-103, -107, -122, -188a, -199a-5p, -205, -340-5p, -345-3p, -452-5p, -499, -878-3p) were commonly found in the three tools utilized and seven miRNAs (miR-9-5p, miR-511-3p, miR-7b-5p, miR-30e-5p, miR-17-5p, miR-122-5p and miR-541-5p) targeting six genes (Traf3, Rock2, Rgs8, Atp1b2, Cacna2d1 and Aldoa) were already validated experimentally in mice. The miRNA-mRNA network of the predicted miRNAs with its respective targets revealed the complex interaction within a biological process leading to sperm dysfunction on exposure to F. Our findings not only suggest that the predicted miRs furnish evidence, but also have the potential to serve as non-invasive biomarkers on F-induced sperm dysfunction. Our data contribute towards elucidating the function of miRNAs in the fluoride induced infertility. miRNA molecular pathways in F intoxication will open new avenues on the use of antagomirs in recovering fertility.
Assuntos
Fluoretos/toxicidade , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Simulação por Computador , Perfilação da Expressão Gênica , Masculino , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Fluoride (F) is an environmental contaminant and industrial pollutant. Molecular mechanisms remain unclear in F induced pulmonary toxicity even after numerous studies. Tamarind fruits act as defluoridating agents, but no study was conducted in in vitro systems. Hence, we aimed to assess the ameliorative impact of the tamarind seed coat extract (TSCE) against F toxicity utilizing lung epithelial cells, A549. Cells were exposed to sodium fluoride (NaF-5 mM) alone and in combination with TSCE (750 ng/ml) or Vitamin C (positive control) for 24 h and analyzed for F content, intracellular calcium ([Ca(2+)]i) level, oxidative stress, mitochondrial integrity and apoptotic markers. TSCE treatment prevented the F induced alterations in [Ca(2+)]i overload, F content, oxidant (reactive oxygen species generation, lipid peroxidation, protein carbonyl content and nitric oxide) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase and glutathione) parameters. Further, TSCE modulates F activated changes in mitochondrial membrane potential, permeability transition pore opening, cytochrome-C release, Bax/Bcl-2 ratio, caspase-3 and PARP-1 expressions. In conclusion, our study demonstrated that TSCE as a potential protective agent against F toxicity, which can be utilized as a neutraceutical.
Assuntos
Extratos Vegetais/farmacologia , Sementes/química , Fluoreto de Sódio/toxicidade , Tamarindus , Animais , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fluoreto de Sódio/sangue , Fluoreto de Sódio/farmacocinética , Superóxido Dismutase/metabolismoRESUMO
Several studies have shown that acute fluoride (F(-)) exposure impairs cardiac function, but the molecular mechanism is not clear. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F(-) for 24 h. A significant accumulation of F(-) was found in the serum and myocardium of experimental rats. F(-) treatment causes myocardial necrosis as evident from increased levels of myocardial troponin I, creatine kinase, lactate dehydrogenase and aspartate transaminase. In addition, F(-) induces myocardial oxidative stress via increased reactive oxygen species, lipid peroxidation, protein carbonyl content and nitrate levels along with decreased in the levels of enzymatic (superoxide dismutase 2, catalase, glutathione peroxidase and glutathione s transferase pi class) and non-enzymatic (reduced glutathione) antioxidants. Notably, F(-) triggers myocardial apoptosis through altered Bax/Bcl2 ratio and increased cytochrome c, caspase 3p20 and terminal deoxynucleotidyl transferase dUTP nick end labeled positive cells. An increased cardiac expression of Nox4 and p38α MAPK in F(-) treated rats indicates the oxidative and apoptotic damage. Moreover, ultra-structural changes, histopathological and luxol fast blue staining demonstrates the degree of myocardial damage at subcellular level. Taken together, these findings reveal that acute F(-) exposure causes cardiac impairment by altering the expression of oxidative stress, apoptosis and necrotic markers.
Assuntos
Apoptose/efeitos dos fármacos , Cariostáticos/intoxicação , Intoxicação por Flúor/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fluoreto de Sódio/intoxicação , Administração Oral , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Cariostáticos/administração & dosagem , Cariostáticos/metabolismo , Relação Dose-Resposta a Droga , Eletrocardiografia/efeitos dos fármacos , Intoxicação por Flúor/etiologia , Intoxicação por Flúor/patologia , Intoxicação por Flúor/fisiopatologia , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Coração/fisiopatologia , Masculino , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Necrose , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Oxirredutases/metabolismo , Distribuição Aleatória , Ratos Wistar , Fluoreto de Sódio/administração & dosagem , Fluoreto de Sódio/sangue , Fluoreto de Sódio/metabolismo , Distribuição Tecidual , Toxicocinética , Disfunção Ventricular/etiologiaRESUMO
Fluoride induced hepatotoxicity has been extensively studied in both humans and animals. However, the mechanism underlying in the hepatotoxicity of experimental fluorosis remains obscure. The severity of fluoride toxicity was reduced by oral administration of certain plant derived antioxidants. Caffeic acid (CA) a polyphenolic compound has diverse range of pharmacological activity in the biological system. Therefore, the present study was aimed to investigate the protective mechanism of CA, against fluoride induced hepatotoxicity in rats. The rats were treated with 300 ppm of NaF via drinking water ad libitum alone and in combination with CA at a dose of 50 mg/kg daily for 30 days by oral intubation. CA treatment significantly prevented fluoride induced hepatic damage as evident from the histopathological studies and declined levels of serum fluoride and liver marker enzymes. A significant decrease in the levels of enzymatic (SOD2, CAT, GPx, and GSTpi class) and nonenzymatic (GSH and Vitamin C) antioxidants along with increased ROS, lipid peroxidation, protein carbonyl content, and nitrate levels by fluoride were also prevented in these groups. In addition, CA inhibits fluoride induced apoptosis by altering the Bax and caspase-3p20 expressions. Further, fluoride induced upregulation of Nox4, p38α MAPK, Hsp60, and downregulation of Hsp27 are the indicators for the detection of oxidative damage, apoptosis, and mitochondrial stress was also modulated by CA. These findings reveal that CA supplementation has a protective effect against fluoride induced hepatotoxicity in rats.
Assuntos
Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fluoreto de Sódio/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Ácido Ascórbico/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Catalase/genética , Catalase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fluoreto de Sódio/toxicidade , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Fluoride (F) is a naturally occurring contaminant in the water. F is essential for normal maintenance of teeth and bones. However, prolonged exposure to high concentration of F is found to be deleterious to teeth, bones and other organs. Besides drinking water, F can enter the body through food, dental products, drugs and industrial emission. People living in areas where F contamination is much higher than the expected level, are found to suffer from not only teeth and bone problem but also other systems, including brain and its functions. Since animals respond to the toxic effects of F like human beings, the deleterious effects of F have been produced experimentally in animals in order to determine the mechanism involved in the action of F. The reports indicating the chronic harmful effects of F in teeth, bones, heart, liver, kidneys, gastrointestinal tract, lungs, brain, blood, hormones and biochemical parameters of experimental animals and in in vitro studies have been reviewed in this article. The neurotoxic action of F that produces chiefly learning and memory impairment has also been included. The review also points out the harmful effects of F on reproduction, its teratogenic action and in inducing premature ageing. Finally, the reports indicating a reversal of certain toxicities of F in experimental animals after withdrawal of its exposure has been included.
Assuntos
Fluoretos/efeitos adversos , Fluorose Dentária/patologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Fluorose Dentária/etiologia , Hormônios/metabolismo , Humanos , Lactação/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Testes de Mutagenicidade , Neoplasias/etiologia , Neoplasias/patologia , Reprodução/efeitos dos fármacos , Teratogênese/efeitos dos fármacos , Dente/efeitos dos fármacos , Dente/patologia , Testes de Toxicidade Crônica , Água/análise , Água/química , Abastecimento de Água/análiseRESUMO
The present study is aimed to evaluate the protective effect of ferulic acid (FA) on fluoride-induced oxidative hepatotoxicity in male Wistar rats. Fluoride (25 mg/L) was given orally to induce hepatotoxicity for 12 weeks. Hepatic damage were assessed using status of pathophysiological markers like serum marker enzymes like aspartate transaminase, alanine transaminase, alkaline phosphatase, acid phosphatase, gamma glutamyl transferase, lactate dehydrogenase, bilirubin, lipid profile, total protein content levels, and histopathological studies. Treatment with FA significantly reduced the degree of histological aberrations and rescued lipid peroxidation, as observed from reduced levels of lipid hydroperoxides, nitric oxide, restored levels of enzymic and non-enzymic antioxidants, and total protein content, with a concomitant decline in the levels of marker enzymes and lipid profile in fluoride-induced rats. These results suggest that ferulic acid has the ability to protect fluoride-induced hepatic damage.